Odyssey CLx Infrared Imaging System Brochure - Digital Version Odyssey Infrared Imaging Systems3....
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QUANTITATIVE WESTERN BLOTS
WIDE, LINEAR DYNAMIC RANGE
NO FILM OR DARKROOM
WIDE RANGE OF APPLICATIONS
The Standard for Western Blot Technology As a researcher, your goal is to efficiently present the most accurate data possible. For more than 30 years, traditional chemiluminescent detection with film has provided data that have been published by scientists worldwide.
Over the past decade, LI-COR® has provided infrared (IR) fluorescent technology with optimized reagents for IR laser- based instrumentation to revolutionize methods for pro- tein detection. This technology has become the standard for quantitative Western blots and eliminated the need for film. Traditional chemiluminescent detection with film provides proven sensitivity, and LI-COR offers industry-leading tech- nology to maintain that sensitivity and improve your data quality to make it the clearest and most accurate it can be. LI-COR has used its expertise in optical design to provide methods for both chemiluminescent, as well as infrared fluo- rescence protein detection, without the use of film.
Benefits of Digital Imaging with the Odyssey CLx:
Save valuable money on film and substrates (Table 1)
Eliminate costs related to darkroom maintenance
No need for multiple exposures
A wide, linear dynamic range without saturation or “blown out” lanes
Accurate detection of strong and weak bands in one exposure
Sensitivity equal to or greater than that of film
Eliminate the need for excessive washes and hazardous waste associated with film development
Reduce the negative environmental impact related to film development. For more information, please visit: www.licor.com/green
Whether you are looking to improve your Western blot data by simply moving to digital chemiluminescent detection or by transitioning to quantitative Western blot technology using infrared fluorescent imaging, we will help you find the best solution for you and your lab.
Quantitative Western Blots The accuracy and linearity of infrared fluorescent
detection provides confidence in differences of protein expression
High Sensitivity Infrared laser technology offers the best
detection at the optimum wavelengths
Wide, Linear Dynamic Range A broad, linear dynamic range accurately detects
both strong and weak bands on the same blot, without the uncertainty and inconvenience of
No Film or Darkroom Save valuable time and money on film and darkroom expenses. Eliminate “blown out” lanes and the need
for multiple exposures
Wide Range of Applications Infrared Western Blots, In-Cell Western™ Assays, On-
Cell Western Assays, Coomassie-Stained Gels, DNA Gels, Protein Arrays, EMSAs, Tissue Section Imaging,
Whole Organ Imaging, Small Animal Imaging, ELISAs
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ODYSSEY® CLx Infrared Imaging System
The Infrared Fluorescence Advantage Advancing Western Blots with Infrared Fluorescence Detection
Cy3 (550 nm)
Cy5 (649 nm)
LI-COR IRDye® 680LT (700 nm)
LI-COR IRDye 800CW (800 nm)
Visible Fluorophores Infrared FluorophoresVisible Fluorophores Infrared Fluorophores
Figure 1. Serial dilutions (10 ng to < 1 pg) of purified human transferrin (Tf) were used to assess Western blot sensitivity. An example of typical results obtained with Odyssey imaging technology. The above data illustrate the detection of 1.2 pg of Tf, while only 4.9-9.5 pg is detected with chemiluminescence. Infrared fluorescent detection sensitivity is approximately 200-fold greater than other studies with visible fluorophores (Cy®3, Cy®5, or FITC). (Data generated on Odyssey Classic)
LI-COR® pioneered infrared Western blots more than ten years ago. Using infrared detection offers numerous benefits, when used with corresponding infrared fluorescent-labeled second- ary antibodies.
Quantitative analysis and a wide linear dynamic range that is not available with traditional chemiluminescent methods
Detect strong and weak bands on the same blot, without blowouts or hidden bands (Fig. 1)
Detect two targets simultaneously on the same membrane to increase quantification accuracy
At the 700 nm and 800 nm infrared wavelengths, both autofluorescence and light scatter are dramatically reduced (Fig. 4)
Infrared dyes offer advanced signal stability that allows for convenient and reproducible data that are not time-sensitive – data are not contingent on the lifespan of an enzymatic reaction
More than 4,000 peer-reviewed publications cite data from Odyssey® Imaging Systems
Figure 2. A dot blot assay was used to compare the linear ranges of chemiluminescent and infrared fluorescent detection. Dilutions of mouse antibody were spotted and detected with HRP- or IRDye® infrared dye-labeled goat anti-mouse antibodies. Chemiluminescent data (Panel A) were col- lected using ECL substrate and a CCD camera with varying exposure times; the infrared image (Panel B) was obtained in a single scan with Odyssey infrared imaging technology. For a 30-minute chemiluminescent exposure, the data set was linear over a 250-fold dynamic range, but not proportional. By contrast, infrared detection displayed a quantitative linear range greater than 4000-fold (3.6 orders of magnitude). A paper detailing this study can be downloaded at www.licor.com/chemiccd. (Data generated on Odyssey Classic)
0.001 0.01 0.1 1 10
30 min 10 min
5 min 1 min
Odyssey Infrared Detection
1 0.0001 0.001 0.01 0.1 101
The dynamic nature of enzyme labels and film allows you to capture only a snapshot of the enzymatic reaction and is highly dependent on timing and exposure, limiting linear range and offering only partially quantitative results (Fig. 2). With the infra- red fluorescence method, film and enzyme labels are replaced with infrared fluorescent-labeled antibodies.
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Sensitivity The Odyssey CLx platform uses infrared laser excitation that out-performs LED and visible white light systems (Fig. 3) for Western blots. Biological materials, membranes, and plastics produce high background due to light scattering and autofluorescence in the visible wavelength range used by most fluorescent imagers. This limits the sensitivity of visible fluorescent systems and makes it difficult to detect low-abundance proteins at endogenous levels without satu-
ration of stronger bands. Infrared laser excitation results in the highest signal-to-noise ratios, and the best detection sensitivity available with a fluorescent system.
Infrared lasers offer high-speed detection with increased sen- sitivity when compared to systems that use LED and visible white light. This increased sensitivity offers a clear image of your data that is unmatched by other digital imagig systems.
A Excitation Light Quality
B Excitation Light Leakage
C Signal-to-Noise Ratio
Figure 3. Improved performance using infrared lasers. A) Infrared lasers deliver excitation light in the narrow wavelength band desired, unlike LED or white light sources. B) Leakage of excitation light (yellow shading) increases image background. C) LI-COR filtering technology dramatically reduces excitation light leakage, for decreased image background and sensitive detection of low abundant targets.
LI-COR’s laser technology has the best detection at the optimum wavelengths. When compared to other instruments using infrared fluorescence, the 700 nm and 800 nm channels within the Odyssey family of imagers are always the most sensitive.
Membrane Autofluorescence is Dramatically Reduced
0 800 nm700 nm532 nm 635 nm
Figure 4. Nitrocellulose and PVDF membranes were imaged with Odyssey infrared imaging technol- ogy at Intensity = 5 for both 700 nm and 800 nm wavelengths. The same membranes were scanned at a 532 nm and 635 nm wave- length with a PMT = 500 on a GenePix® 4100A (Molecular Devices). Autofluorescence was much lower at infrared wavelengths.
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